Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins
50 Pages Posted: 21 Nov 2018 Sneak Peek Status: Under ReviewMore...
Engineering proteins to assembly into user-defined structures is key in their development for biotechnological applications. However, designing generic rather than bespoke solutions is a challenging task. Here, we describe an expandable recombinant assembly system that produces scalable protein cages via split intein-mediated native chemical ligation. Three types of construct are used: a pair of oligomeric mirror-image “half-cages” and an extendable “linker". All constructs are composed of modular protein domains chosen to fulfil the required geometries, fused to two orthogonal pairs of split-intein halves to drive assembly when mixed. This combination enables both one-pot construction of two-component cages and stepwise assembly of larger three-component scalable cages. To illustrate the system’s versatility, tetrahedral half-cages and linker constructs comprising consensus-designed repeat proteins were ligated in one-pot and stepwise reactions. Under mild conditions rapid high yielding ligations were obtained, from which discrete proteins cages were easily purified and shown to form the desired trigonal bipyramidal structures.
Keywords: directed protein assembly, native chemical ligation, protein conjugation, split-intein ligation, protein design, protein semi-synthesis
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